The invention relates to relatively short peptides (termed xcfx84-conotoxins herein), about 10-20 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include two disulfide bonds.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
The predatory cone snails (Conus) have developed a unique biological strategy. Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms. Many of these peptides are among the smallest nucleic acid-encoded translation products having defined conformations, and as such, they are somewhat unusual. Peptides in this size range normally equilibrate among many conformations. Proteins having a fixed conformation are generally much larger.
The cone snails that produce these peptides are a large genus of venomous gastropods comprising approximately 500 species. All cone snail species are predators that inject venom to capture prey, and the spectrum of animals that the genus as a whole can envenomate is broad. A wide variety of hunting strategies are used, however, every Conus species uses fundamentally the same basic pattern of envenomation.
Several peptides isolated from Conus venoms have been characterized. These include the xcex1-, xcexc- and xcfx89-conotoxins which target nicotinic acetylcholine receptors, muscle sodium channels, and neuronal calcium channels, respectively (Olivera et at., 1985). Conopressins, which are vasopressin analogs, have also been identified (Cruz et al. 1987). In addition, peptides named conantokins have been isolated from Conus geographus and Conus tulipa (Mena et al., 1990; Haack et al., 1990).
Chronic or intractable pain, which may result from degenerative conditions or debilitating diseases, is currently treated with a variety of analgesic compounds, often opioid compounds such as morphine. Likewise, neuropathic pain, typically a chronic condition attributable to injury or partial transection of a peripheral nerve, is also conventionally treated with opioid compounds such as morphine.
Conventional therapies for pain produce analgesiaxe2x80x94a loss of sensitivity to pain without the loss of consciousness. Opioid compounds have been used widely to produce analgesia, including plant-derived opioids such as morphine, and endogenous opioids such as met- and leu-enkephalins, as well as beta-endorphin.
Opioid compounds, while effective in producing analgesia for many types of pain, may induce tolerance in some patients. When a patient becomes tolerant, increasing doses of the opioid are required to produce the desired analgesic effect. In addition, these compounds frequently result in a physical dependence in patients, and may have side effects at high doses.
The analgesic effects and adverse actions of various N-methyl-D-aspartate (NMDA) receptor antagonists has been shown to vary depending on the site of action and potency of the drug. For example, NMDA receptor antagonists acting at the ion channel in a noncompetitive manner (e.g., MK-801 and phenylcyclidine (PCP)) or competitive inhibitors, show analgesic activity but show motor impairment at equivalent doses. Glycine B-site NMDA antagonists appear to have analgesic activity at doses that do not impair motor function. Conantokins, which are polyamine-site NMDA antagonist compounds have analgesic effects at doses which do not produce overt side effects (PCT published application WO 98/03189).
It is desired to provide additional compounds which have analgesic properties.
The invention relates to relatively short peptides (termed xcfx84-conotoxins herein), about 10-25 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include two disulfide bonds.
More specifically, the present invention is directed to xcfx84-conotoxin peptides having the general formula I:
Xaa1-Xaa2-Xaa3-Xaa4-Cys-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Cys-Cys-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19 (SEQ ID NO:1), wherein Xaa1 is des-Xaa1, Asp, Glu or xcex3-carboxy-Glu (Gla); Xaa2 is des-Xaa2, Gln, Asn, Glu, Trp (D or L), neo-Trp, halo-Trp or any unnatural aromatic amino acid; Xaa3 is des-Xaa3, Gly, Ala, Asn or Gln; Xaa4 is des-Xaa4, Val, Leu (D or L), Ile, Ala, Gly, Glu, Gla, Asp, Ser, Thr, Phe, Trp (D or L), neo-Trp, halo-Trp (D or L) or any unnatural aromatic amino acid; Xaa5 is Pro, hydroxy-Pro, Gln, Asn, Glu, Gla, Ala, Gly, Lys, Arg, Ile, Val, homoarginine, ornithine, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid; Xaa6 is Val, Phe, Thr, Ser, Glu, Gla, Asp, Asn, Gln, Ala, Gly, Ile, Leu (D or L) Met, Pro, hydroxy-Pro, Arg, homoarginine, ornithine, Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, any unnatural basic amino acid or any unnatural aromatic amino acid; Xaa7 is any Val, Ile, Asn, Leu (D or L), Gln, Gly, Ala, Phe, Glu, Gla, Arg, ornithine, homoarginine, Lys, N-methy-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, any unnatural basic amino acid or any unnatural aromatic amino acid; Xaa8 is Ile, Leu (D or L), Met, Thr, Ser, Pro, hydroxy-Pro, Gln, Asp, Glu, Gla, Asn, Arg, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, any unnatural basic amino acid, any unnatural aromatic amino acid or any unnatural hydroxy containing amino acid; Xaa1 is des-Xaa9, Ala, Gly, Asp, Glu, Gla, Trp (D or L) neo-Trp, halo-Trp (D or L), Lys, N-methy-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, homoarginine, ornithine, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any unnatural basic amino acid; Xaa10 is des-Xaa10, Ile, Leu (D or L), Val, Glu, Gla, Asp, Thr, Ser, Pro, hydroxy-Pro, Trp (D or L), neo-Trp, halo-Trp (D or L), Phe, any unnatural aromatic amino acid or any unnatural hydroxy containing amino acid; Xaa1, is des-Xaa11, Gln, Asn, Leu (D or L), Ile, Val, Ala, Gly, Trp (D or L), neo-Trp, halo-Trp (D or L), Arg, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, any unnatural basic amino acid or any unnatural aromatic amino acid; Xaa12 is des-Xaa12, Ala, Gly, Phe, Trp (D or L), neo-Trp, halo-Trp (D or L) or any unnatural aromatic amino acid; Xaa13 is des-Xaa13, Glu, Gla, Asp, Phe or any unnatural aromatic amino acid; Xaa14 is des-Xaa14, Ile, Val or Leu (D or L); Xaa15 is des-Xaa15, Thr, Ser, Arg, homoarginine, ornithine, Lys, N-methy-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid; Xaa16 is des-Xaa16, Glu, Gla or Asp; Xaa17 is des-Xaa17, Asn or Gln; Xaa18 is des-Xaa18, Asp, Glu or Gla; Xaa19 is des-Xaa19, Phe or any unnatural aromatic amino acid. The C-terminus may contain a free carboxyl group or an amide group. The halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr and bromine for Trp. The Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L). The Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O-phospho-derivatives. The acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala.
The present invention is also directed to novel specific xcfx84-conotoxin peptides of general formula I having the formulas:
Phe-Cys-Cys-Xaa1-Val-Ile-Arg-Xaa2-Cys-Cys-Xaa3 (SEQ ID NO:2);
Phe-Cys-Cys-Xaa1-Phe-Ile-Arg-Xaa2-Cys-Cys-Xaa3 (SEQ ID NO:3);
Cys-Cys-Gln-Thr-Phe-Xaa2-Xaa3-Cys-Cys-Gln (SEQ ID NO:4);
Xaa4-Gly-Xaa3-Cys-Cys-Xaa5-Xaa6-Asn-Ile-Ala-Cys-Cys-Ile (SEQ ID NO:5);
Gly-Cys-Cys-Ala-Arg-Leu-Thr-Cys-Cys-Val (SEQ ID NO:6);
Asn-Gly-Cys-Cys-Xaa1-Xaa5-Gln-Met-Arg-Cys-Cys-Thr (SEQ ID NO:7);
Asp-Xaa3-Asn-Ser-Cys-Cys-Gly-Xaa6-Asn-Xaa1-Gly-Cys-Cys-Xaa1-Xaa3 (SEQ ID NO:8);
Xaa4-Gly-Xaa3-Cys-Cys-Xaa5-Xaa6-Asn-Ile-Arg-Cys-Cys-Val (SEQ ID NO:9);
Xaa6-Cys-Cys-Xaa6-Asp-Gly-Xaa3-Cys-Cys-Thr-Ala-Ala-Xaa1-Leu-Thr (SEQ ID NO:10);
Gly-Cys-Cys-Xaa6-Asp-Gly-Xaa3-Cys-Cys-Thr-Ala-Ala-Xaa1-Leu-Thr(SEQ ID NO:11);
Asn-Gly-Cys-Cys-Arg-Ala-Gly-Asp-Cys-Cys-Ser-Arg-Phe-Xaa6-Ile-Xaa5-Xaa6-Asn-Asp-Phe (SEQ ID NO:12);
Asn-Ala-Cys-Cys-Ile-Val-Arg-Gln-Cys-Cys (SEQ ID NO:13);
Asn-Gly-Cys-Cys-Arg-Ala-Gly-Asp-Cys-Cys-Ser (SEQ ID NO:14);
Cys-Cys-Xaa1-Arg-Arg-Leu-Ala-Cys-Cys-Ile-lie (SEQ ID NO:15);
Cys-Cys-Xaa1-Asn-Xaa5-Xaa1-Cys-Cys-Phe-Ile (SEQ ID NO:16);
Gly-Cys-Cys-Ala-Met-Leu-Thr-Cys-Cys-Val (SEQ ID NO:17);
Leu-Cys-Cys-Val-Thr-Xaa6-Asp-Xaa3-Cys-Cys-Xaa6-Xaa3-Xaa3 (SEQ ID NO:18); and
Val-Cys-Cys-Arg-Xaa1-Val-Gln-Asp-Cys-Cys-Ser (SEQ ID NO:19);
wherein Xaa1 is Pro or hydroxy-Pro; Xaa2 is Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa3 is Trp or halo-Trp; Xaa4 is Gln or pyro-Glu; Xaa5 is Lys, N-methyl-Lys, N,N-dimethyl-Lys or N,n,N-trimethyl-Lys, Xaa6 is Glu or gamma-carboxy-Glu (Gla); and the C-terminus contains a carboxyl or amide group. The halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr and bromine for Trp. In addition, the Arg residues may be substituted by Lys, ornithine, homoargine, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid; the Lys residues may be substituted by Arg, ornithine, homoargine, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid; the Tyr residues may be substituted with any unnatural hydroxy containing amino acid; the Ser residues may be substituted with Thr; the Thr residues may be substituted with Ser; and the Phe and Trp residues may be substituted with any unnatural aromatic amino acid. The Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L). The Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O-phospho-derivatives. The acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala.
More specifically, the present invention is directed to the following xcfx84-conotoxin peptides of general formula I:
AuVA: SEQ ID NO:2, wherein Xaa1 is Pro, Xaa2 is Tyr and Xaa3 is Trp;
AuVB: SEQ ID NO:3, wherein Xaa1 is Pro, Xaa2 is Tyr and Xaa3 is Trp;
Tx5.1: SEQ ID NO:4, wherein Xaa2 is Tyr and Xaa3 is Trp;
G5.1: SEQ ID NO:5, wherein Xaa3 is Trp, Xaa4 is Gln, Xaa5 is Lys and Xaa6is Glu;
Qc5.1: SEQ ID NO:6;
PVA: SEQ ID NO:7, wherein Xaa1 is Pro and Xaa5 is Lys;
Im5.1: SEQ ID NO:8, wherein Xaa1 is Pro, Xaa3 is Trp and Xaa1 is Lys;
G5.2: SEQ ID NO:9, wherein Xaa3 is Trp, Xaa4 is Gln, Xaa5 is Lys and Xaa6 is Glu;
Tx5.2a: SEQ ID NO:10, wherein Xaa1 is Pro, Xaa3 is Trp and Xaa6 is Glu;
Tx5.2b: SEQ ID NO:11, wherein Xaa1 is Pro, Xaa3 is Trp and Xaa6 is Glu;
Mr5.1: SEQ ID NO:12, wherein Xaa1 is Lys and Xaa6 is Glu;
Mr5.2: SEQ ID NO:13;
Mr5.3: SEQ ID NO:14;
Ca5.1: SEQ ID NO:15, wherein Xaa1 is Pro;
Ca5.2: SEQ ID NO:16, wherein Xaa1 is Pro and Xaa5 is Lys;
Qc5.2: SEQ ID NO:17;
Gm5.1: SEQ ID NO:18, wherein Xaa3 is Trp and Xaa6 is Glu; and
Gm5.2: SEQ ID NO:19, wherein Xaa1 is Pro.
The C-terminus preferably contains a carboxyl group for the peptides AuVA, AuVB, G5.1, PVA, G5.2, Mr5.2, Mr5.3 and Gm5.1 The C-terminus of the other peptides preferably contains an amide group.
Examples of unnatural aromatic amino acid include, but are not limited to, such as nitro-Phe, 4-substituted-Phe wherein the substituent is C1-C3 alkyl, carboxyl, hyrdroxymethyl, sulphomethyl, halo, phenyl, xe2x80x94CHO, xe2x80x94CN, xe2x80x94SO3H and xe2x80x94NHAc. Examples of unnatural hydroxy containing amino acid, include, but are not limited to, such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr. Examples of unnatural basic amino acids include, but are not limited to, N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala, 2-[3-(2S)pyrrolininyl)-Gly and 2-[3-(2S)pyrrolininyl)-Ala. These and other unnatural basic amino acids, unnatural hydroxy containing amino acids or unnatural aromatic amino acids are described in Building Block Index, Version 3.0 (1999 Catalog, pages 4-47 for hydroxy containing amino acids and aromatic amino acids and pages 66-87 for basic amino acids), incorporated herein by reference, by and available from RSP Amino Acid Analogues, Inc., Worcester, Mass. Examples of synthetic acid amino acids include those derivatives bearing acidic functionality, including carboxyl, phosphate, sulfonate and synthetic tetrazolyl derivatives such as described by Ornstein et al. (1993) and in U.S. Pat. No. 5,331,001, each incorporated herein by reference.
Optionally, in the peptides of general formula I and the specific peptides described above, the Asn residues may be modified to contain an N-glycan and the Ser and Thr residues may be modified to contain an O-glycan. In accordance with the present invention, a glycan shall mean any N-, S- or O-linked mono-, di-, tri-, poly- or oligosaccharide that can be attached to any hydroxy, amino or thiol group of natural or modified amino acids by synthetic or enzymatic methodologies known in the art. The monosaccharides making up the glycan can include D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GIcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose. These saccharides may be structurally modified, e.g., with one or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid, including combinations thereof. The glycan may also include similar polyhydroxy groups, such as D-penicillamine 2,5 and halogenated derivatives thereof or polypropylene glycol derivatives. The glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3. The linkage between the glycan and the amino acid may be alpha or beta, preferably alpha and is 1xe2x88x92.
Core O-glycans have been described by Van de Steen et al. (1998), incorporated herein by reference. Mucin type O-linked oligosaccharides are attached to Ser or Thr (or other hydroxylated residues of the present peptides) by a GalNAc residue. The monosaccharide building blocks and the linkage attached to this first GalNAc residue define the xe2x80x9ccore glycans,xe2x80x9d of which eight have been identified. The type of glycosidic linkage (orientation and connectivities) are defined for each core glycan. Suitable glycans and glycan analogs are described further in U.S. patent application Ser. No. 09/420,797, filed Oct. 19 , 1999 and in PCT Application No. PCT/US99/24380, filed Oct. 19, 1999, both incorporated herein by reference. A preferred glycan is Gal(xcex21xe2x86x923)GalNAc(xcex11xe2x86x92).
Optionally, in the peptides of general formulas I and II and the specific peptides described above, pairs of Cys residues may be replaced pairwise with isosteric lactam or ester-thioether replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp) or Cys/Ala combinations. Sequential coupling by known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et al., 1997) allows replacement of native Cys bridges with lactam bridges. Thioether analogs may be readily synthesized using halo-Ala residues commercially available from RSP Amino Acid Analogues.
The present invention is further directed to propeptides and nucleic acid sequences encoding the propeptides or peptides as described in further detail herein.
SEQ ID NO:1is generic formula I for xcfx84-conotoxin peptides. SEQ ID NO:2 is a generic formula for the peptide AuVA. SEQ ID NO:3 is a generic formula for the peptide AuVB. SEQ ID NO:4 is a generic formula for the peptide Tx5.1. SEQ ID NO:5 is a generic formula for the peptide G5.1. SEQ ID NO:6 is a generic formula for the peptide Qc5.1. SEQ ID NO:7 is a generic formula for the peptide PVA. SEQ ID NO:8 is a generic formula for the peptide Im5.1. SEQ ID NO:9 is a generic sequence for the peptide G5.2. SEQ ID NO:10 is a generic sequence for the peptide Tx5.2a. SEQ ID NO:11 is a generic sequence for the peptide Tx5.2b. SEQ ID NO:12 is a generic sequence for the peptide Mr5.1. SEQ ID NO:13 is a generic sequence for the peptide Mr5.2. SEQ ID NO:14 is a generic formula for the peptide Mr5.3. SEQ ID NO:15 is a generic formula for the peptide Ca5.1. SEQ ID NO:16 is a generic formula for the peptide Ca5.2. SEQ ID NO:17 is a generic formula for the peptide Qc5.2. SEQ ID NO:18 is a generic formula for the peptide Gm5.1. SEQ ID NO:19 is a generic formula for the peptide Gm5.2. SEQ ID NO:20 is a DNA sequence coding for the Tx5.1 propeptide. SEQ ID NO:21 is the amino acid sequence of the Tx5.1 propeptide. SEQ ID NO:22 is a DNA sequence coding for the G5.1 propeptide. SEQ ID NO:23 is the amino acid sequence of the G5.1 propeptide. SEQ ID NO:24 is a DNA sequence coding for the Qc5.1 propeptide. SEQ ID NO:25 is the amino acid sequence of the Qc5.1 propeptide. SEQ ID NO:26 is a DNA sequence coding for the Im5.1 propeptide. SEQ ID NO:27 is the amino acid sequence of the Im5.1 propeptide. SEQ ID NO:28 is a DNA sequence coding for the G5.2 propeptide. SEQ ID NO:29 is the amino acid sequence of the G5.2 propeptide. SEQ ID NO:30 is a DNA sequence coding for the Tx5.2 propeptide. SEQ ID NO:31 is the amino acid sequence of the Tx5.2 propeptide. SEQ ID NO:32 is a DNA sequence coding for the Tx5.3 propeptide. SEQ ID NO:33 is the amino acid sequence of the Tx5.3 propeptide. SEQ ID NO:34 is a DNA sequence coding for the Mr5.1 peptide. SEQ ID NO:35 is the amino acid sequence of the Mr5.1 peptide. SEQ ID NO:36 is a DNA sequence coding for the Mr5.2 peptide. SEQ ID NO:37 is the amino acid sequence of the Mr5.2 peptide. SEQ ID NO:38 is a DNA sequence coding for the Mr5.3 propeptide. SEQ ID NO:39 is the amino acid sequence of the Mr5.3 propeptide. SEQ ID NO:40 is a DNA sequence coding for the Ca5.1 propeptide. SEQ ID NO:41 is the amino acid sequence of the Ca5.1 propeptide. SEQ ID NO:42 is a DNA sequence coding for the Ca5.2 propeptide. SEQ ID NO:43 is the amino acid sequence of the Ca5.2 propeptide. SEQ ID NO:44 is a DNA sequence coding for the Qc5.2 propeptide. SEQ ID NO:45 is the amino acid sequence of the Qc5.2 propeptide. SEQ ID NO:46 is a DNA sequence coding for the Gm5.1 propeptide. SEQ ID NO:47 is the amino acid sequence of the Gm5.1 propeptide. SEQ ID NO:48 is a DNA sequence coding for the Gm5.2 propeptide. SEQ ID NO:49 is the amino acid sequence of the Gm5.2 propeptide.
The invention relates to relatively short peptides (termed r-conotoxins herein), about 10-25 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include two disulfide bonds.
The present invention, in another aspect, relates to a pharmaceutical composition comprising an effective amount of an xcfx84-conotoxin peptide, a mutein thereof, an analog thereof, an active fragment thereof or pharmaceutically acceptable salts . Such a pharmaceutical composition has the capability of acting as an antagonist for acetylcholine receptors and as analgesic agents for the treatment of pain, including migraine. Thus, the pharmaceutical compositions of the present invention are useful in the treatment of pain (whether acute or chronic), including chronic pain, and neuropathic pain, without undesirable side effects.
The xcfx84-conotoxin peptides described herein are sufficiently small to be chemically synthesized. General chemical syntheses for preparing the foregoing xcfx84-conotoxin peptides are described hereinafter. Various ones of the xcfx84-conotoxin peptides can also be obtained by isolation and purification from specific Conus species using the technique described in U.S. Pat. Nos. 4,447,356 (Olivera et al., 1984); 5,514,774; 5,719,264; and 5,591,821, as well as in PCT published application WO 98/03189, the disclosures of which are incorporated herein by reference.
Although the xcfx84-conotoxin peptides of the present invention can be obtained by purification from cone snails, because the amounts of xcfx84-conotoxin peptides obtainable from individual snails are very small, the desired substantially pure xcfx84-conotoxin peptides are best practically obtained in commercially valuable amounts by chemical synthesis using solid-phase strategy. For example, the yield from a single cone snail may be about 10 micrograms or less of xcfx84-conotoxin peptide. By xe2x80x9csubstantially purexe2x80x9d is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% purity and preferably at least about 95% purity. Chemical synthesis of biologically active xcfx84-conotoxin peptides depends of course upon correct determination of the amino acid sequence.
The xcfx84-conotoxin peptides can also be produced by recombinant DNA techniques well known in the art. Such techniques are described by Sambrook et al. (1989). A gene of interest (i.e., a gene that encodes a suitable xcfx84-conotoxin peptide) can be inserted into a cloning site of a suitable expression vector by using standard techniques. These techniques are well known to those skilled in the art. The expression vector containing the gene of interest may then be used to transfect the desired cell line. Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection or electroporation may be utilized. A wide variety of host/expression vector combinations may be used to express a gene encoding a conotoxin peptide of interest. Such combinations are well known to a skilled artisan. The peptides produced in this manner are isolated, reduced if necessary, and oxidized to form the correct disulfide bonds.
One method of forming disulfide bonds in the xcfx84-conotoxin peptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures or at room temperature. This procedure results in the creation of a substantial amount of the bioactive, disulfide-linked peptides. The oxidized peptides are fractionated using reverse-phase high performance liquid chromatography (HPLC) or the like, to separate peptides having different linked configurations. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined. However, because of the dilution resulting from the presence of other fractions of less biopotency, a somewhat higher dosage may be required.
The peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
In conventional solution phase peptide synthesis, the peptide chain can be prepared by a series of coupling reactions in which constituent amino acids are added to the growing peptide chain in the desired sequence. Use of various coupling reagents, e.g., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole, various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide, and the various cleavage reagents, to carry out reaction in solution, with subsequent isolation and purification of intermediates, is well known classical peptide methodology. Classical solution synthesis is described in detail in the treatise, xe2x80x9cMethoden der Organischen Chemie (Houben-Weyl): Synthese von Peptiden,xe2x80x9d (1974). Techniques of exclusively solid-phase synthesis are set forth in the textbook, xe2x80x9cSolid-Phase Peptide Synthesis,xe2x80x9d (Stewart and Young, 1969), and are exemplified by the disclosure of U.S. Pat. No. 4,105,603 (Vale et al., 1978). The fragment condensation method of synthesis is exemplified in U.S. Pat. No. 3,972,859 (1976). Other available syntheses are exemplified by U.S. Pat. Nos. 3,842,067 (1974) and 3,862,925 (1975). The synthesis of peptides containing xcex3-carboxyglutamic acid residues is exemplified by Rivier et al. (1987), Nishiuchi et al. (1993) and Zhou et al. (1996).
Common to such chemical syntheses is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical reaction from occurring at that site until the group is ultimately removed. Usually also common is the protection of an xcex1-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the xcex1-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that, as a step in such a synthesis, an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with appropriate side-chain protecting groups linked to various ones of the residues having labile side chains.
As far as the selection of a side chain amino protecting group is concerned, generally one is chosen which is not removed during deprotection of the xcex1-amino groups during the synthesis. However, for some amino acids, e.g., His, protection is not generally necessary. In selecting a particular side chain protecting group to be used in the synthesis of the peptides, the following general rules are followed: (a) the protecting group preferably retains its protecting properties and is not split off under coupling conditions, (b) the protecting group should be stable under the reaction conditions selected for removing the xcex1-amino protecting group at each step of the synthesis, and (c) the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
It should be possible to prepare many, or even all, of these peptides using recombinant DNA technology. However, when peptides are not so prepared, they are preferably prepared using the Merrifield solid-phase synthesis, although other equivalent chemical syntheses known in the art can also be used as previously mentioned. Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected xcex1-amino acid to a suitable resin. Such a starting material can be prepared by attaching an xcex1-amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine (MBHA) resin. Preparation of the hydroxymethyl resin is described by Bodansky et al. (1966). Chloromethylated resins are commercially available from Bio Rad Laboratories (Richmond, Calif.) and from Lab. Systems, Inc. The preparation of such a resin is described by Stewart and Young (1969). BHA and MBHA resin supports are commercially available, and are generally used when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus. Thus, solid resin supports may be any of those known in the art, such as one having the formulae xe2x80x94Oxe2x80x94CH2-resin support, xe2x80x94NH BHA resin support, or xe2x80x94NHxe2x80x94MBHA resin support. When the unsubstituted amide is desired, use of a BHA or MBHA resin-is preferred, because cleavage directly gives the amide. In case the N-methyl amide is desired, it can be generated from an N-methyl BHA resin. Should other substituted amides be desired, the teaching of U.S. Pat. No. 4,569,967 (Kornreich et al., 1986) can be used, or should still other groups than the free acid be desired at the C-terminus, it may be preferable to synthesize the peptide using classical methods as set forth in the Houben-Weyl text (1974).
The C-terminal amino acid, protected by Boc or Fmoc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in K. Horiki et al. (1978), using KF in DMF at about 60xc2x0 C. for 24 hours with stirring, when a peptide having free acid at the C-terminus is to be synthesized. Following the coupling of the BOC-protected amino acid to the resin support, the xcex1-amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone. The deprotection is carried out at a temperature between about 0xc2x0 C. and room temperature. Other standard cleaving reagents, such as HCl in dioxane, and conditions for removal of specific xcex1-amino protecting groups may be used as described in Schroder and Lubke (1965).
After removal of the xcex1-amino-protecting group, the remaining xcex1-amino- and side chain-protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor. Selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as a coupling reagent is N,Nxe2x80x2-dicyclohexylcarbodiimide (dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIC), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(Benzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium tetrafluoroborate (TBTU) in the presence of HoBt or HoAt).
The activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art. Examples of suitable activating reagents are carbodiimides, such as N,Nxe2x80x2-diisopropylcarbodiimide and N-ethyl-Nxe2x80x2-(3-dimethylaminopropyl)carbodiimide. Other activating reagents and their use in peptide coupling are described by Schroder and Lubke (1965) and Kapoor (1970).
Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide (DMF):CH2Cl2 (1:1) or in DMF or CH2Cl2 alone. In cases where intermediate coupling occurs, the coupling procedure is repeated before removal of the xcex1-amino protecting group prior to the coupling of the next amino acid. The success of the coupling reaction at each stage of the synthesis, if performed manually, is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970). Coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al. (1978).
After the desired amino acid sequence has been completed, the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry), which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and also the xcex1-amino protecting group at the N-terminus if it was not previously removed to obtain the peptide in the form of the free acid. If Met is present in the sequence, the Boc protecting group is preferably first removed using trifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential S-alkylation. When using hydrogen fluoride or TFA for cleaving, one or more scavengers such as anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the reaction vessel.
Cyclization of the linear peptide is preferably affected, as opposed to cyclizing the peptide while a part of the peptido-resin, to create bonds between Cys residues. To effect such a disulfide cyclizing linkage, fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, which is thereafter suitably cyclized and deprotected. Alternatively, deprotection, as well as cleavage of the peptide from the above resins or a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), can take place at 0xc2x0 C. with hydrofluoric acid (HF) or TFA, followed by oxidation as described above.
The peptides are also synthesized using an automatic synthesizer. Amino acids are sequentially coupled to an MBHA Rink resin (typically 100 mg of resin) beginning at the C-terminus using an Advanced Chemtech 357 Automatic Peptide Synthesizer. Couplings are carried out using 1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by 2-(1 H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diethylisopro-pylethylamine (DIEA). The FMOC protecting group is removed by treatment with a 20% solution of piperidine in dimethylformamide(DMF). Resins are subsequently washed with DMF (twice), followed by methanol and NMP.
Muteins, analogs or active fragments, of the foregoing conotoxin peptides are also contemplated here. See, e.g., Hammerland et al, Eur. J. Pharmacol., 226, pp. 239-244 (1992). Derivative muteins, analogs or active fragments of the conotoxin peptides may be synthesized according to known techniques, including conservative amino acid substitutions, such as outlined in U.S. Pat. Nos. 5,545,723 (see particularly col. 2, line 50xe2x80x94col. 3, line 8); 5,534,615 (see particularly col. 19, line 45xe2x80x94col. 22, line 33); and 5,364,769 (see particularly col. 4, line 55xe2x80x94col. 7, line 26), each herein incorporated by reference.
Pharmaceutical compositions containing a compound of the present invention or its pharmaceutically acceptable salts as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington""s Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.). Typically, an antagonistic amount of the active ingredient will be admixed with a pharmaceutically acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral or parenteral. The compositions may further contain antioxidizing agents, stabilizing agents, preservatives and the like. For examples of delivery methods see U.S. Pat. No. 5,844,077, incorporated herein by reference.
For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
For parenteral administration, the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
The active agent is preferably administered in an therapeutically effective amount. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or spealists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington""s Pharmaceutical Sciences. Typically the active agents of the present invention exhibit their effect at a dosage range from about 0.001 mg/kg to about 250 mg/kg, preferably from about 0.01 mg/kg to about 100 mg/kg of the active ingredient, more preferably from a bout 0.05 mg/kg to about 75 mg/kg. A suitable dose can be administered in multiple sub-doses per day. Typically, a dose or sub-dose may contain from about 0.1 mg to about 500 mg of the active ingredient per unit dosage form. A more preferred dosage will contain from about 0.5 mg to about 100 mg of active ingredient per unit dosage form. Dosages are generally initiated at lower levels and increased until desired effects are achieved.
Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
The active agents, which are peptides, can also be administered in a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region. Suitable delivery systems are described in U.S. Pat. No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635. Suitable DNA sequences can be prepared synthetically for each active agent on the basis of the developed sequences and the known genetic code.